β-Glucosidase (EC3. 2. 1, 21) belongs to the class of cellulose hydrolases and is one kind of enzyme, which can used for hydrolysis or transferring β-1,4-glycosidic bonds. The β-glucosidase is an important constituent in cellulase system. No less than three kinds of enzymes, namely endoglucanase, cellobiohydrolase and β-glucosidase are needed for the cellulose conversion into glucose. Cellulose can be degraded into cellobiose by endoglucanase and cellobiohydrolase, and further decomposed into glucose by β-glucosidase. Releasing of glucose from cellobiose is the key rate-limiting step for the cellulose hydrolysis.
However, the content of β-glucosidase in cellulase system is very low, which is less than 1%, and the activity is generally relatively low, which is a main bottleneck for cellulose enzymatic conversion. For a long time, the poor enzymes properties, low yields of the enzymes and low specific activity of the enzymes are main bottlenecks on their actual application. Although β-glucosidase widely exists in nature, fungi, bacteria and other microbes have low enzyme production efficiency, are difficult to obtain quantities of β-glucosidase, and still have relatively poor thermostability. At present, the activity of β-glucosidase can not meet the needs of industrial production yet, and the cost is further relatively high. In the saccharification process of cellulose, the most appropriate action temperature of the cellulase is about 50° C. generally, while the most appropriate temperature for yeast fermentation is about 30° C. How to coordinate the temperature of the two processes is the key for efficiently producing ethanol by simultaneous saccharification and fermentation (SSF), and one of the methods for solving the contradiction is using a thermophilic yeast instead of traditional yeast. Thus, cloning and expression of β-glucosidase genes have been one of important links on cellulose studies. So far, β-glucosidase genes have been cloned from hundreds of microbes, and many β-glucosidase genes from microbes are heterologously expressed. In recent years, gene engineering technology was used to construct recombinant microorganisms to secret and express β-glucosidases with high activity and thermal stability, which is a research hotspot in the field of cellulose studies.
The known patents and literature reports of thermophilic β-glucosidase and the genes thereof are as follows: β-glucosidase gene fragment BCC2871 from Periconia sp. reported by Piyanun Harnpicharnchai, et al., [Protein Expres Purif, 67: 61-69, 2009]; β-glucosidase gene Cel3a from thermophilic fungal Talaromyces emersonii reported by Patrick Murraya, et al., [Protein Expres Purif, 38: 248-257, 2004]; β-glucosidase gene PtBglu3 from Paecilomyces thermophila reported by Qiaojuan Yan, et al., [Protein Expres Purif, 84: 64-72, 2012]; β-glucosidase gene BglC from Thermobifida Fusca reported by Xiao-Qiong Pei et al., [Bioresour. Technol., 102: 3337-3342, 2011]; and DSM 50691 thermophilic β-glucosidase gene Tt-bgl from Thermotoga thermarum reported by Linguo Zhao, et al., [J Mol Catal B-Enzym, 95: 62-69, 2013] and so on. There are a variety of types of β-glucosidase produced by microbes, and β-glucosidases from different, sources showed different characteristics. Due to different industrial applications, the β-glucosidase with new properties needs to be developed continuously for being better applicable to the industrial production needs. Wherein, thermophilic enzymes have peat advantages, including higher reaction rate, long-time reaction stability low pollution possibility and strengthen the tolerance to chemical reagents, and high enzyme activities under the required special conditions and so on. So the development of thermophilic β-glucosidase has become the research hotspot.
In our previous studies, we obtained two strains of strains producing β-glucosidase: Trichoderma viride W2 with patent number of ZL 201010577713.6 and Hypocrea sp. W63 with patent number of ZL 201110417104.9, both of them showed thermophilic and ethanol-resistant properties. Wherein the optimal pH value of the Trichoderma viride W2 is 4.8, the optimal temperature is 70° C. The activity of β-glucosidase can be largely improved by 1.6 times at 10% (v/v) of ethanol concentration, which is stable below 30% (v/v) of ethanol concentration; and the optimal pH value of Hypocrea sp. W63 is 4.8, the optimal temperature is 65° C., and the concentration of ethanol at 10% (v/v) also has the greatest effect of promoting on enzyme activity, which can improve the enzyme activity of β-glucosidase by nearly 1 time and the highest ethanol resistance concentration also is 30% (v/v).
Under normal circumstances, the cellulose produced by fungi mainly comprises endoglucanase and cellobiohydrolase, and the content of β-glucosidase in the cellulose system is the lowest, which is less than 1%. Many kinds of enzyme produced by gene engineering strain have the advantages of high genetic stability, rapid enzyme production, high enzyme yield and so on, and the produced recombinant enzymes are intended to meet the demands of future industrial applications. According to worldwide reports, the gene cloning of thermophilic β-glucosidases were mainly focused on cloned from thermophilic bacteria and fungi. However, the β-glucosidase produced by the thermophilic microbes do not have thermophilicity and thermal stability necessarily. As for β-glucosidases producing, microbes Trichoderma is one of the species researched most extensively before, but there are no report about β-glucosidase genes with thermophilic ethanol-resistant performance, which are cloned from Hypocrea sp.